Detection and genomic characterization of Klebsiella pneumoniae and Escherichia coli harboring tet(X4) in black kites (Milvus migrans) in Pakistan

The emergence of plasmid-mediated tigecycline resistance gene tet(X4) among clinically relevant bacteria has promoted significant concerns, as tigecycline is considered a last-resort drug against serious infections caused by multidrug-resistant bacteria. We herein focused on the isolation and molecular characterization of tet(X4)-positive Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) in wild bird populations with anthropogenic interaction in Faisalabad, Pakistan. A total of 150 birds including black kites (Milvus migrans) and house crows (Corvus splendens) were screened for the presence of tigecycline resistance K. pneumoniae and E. coli. We found two K. pneumoniae and one E. coli isolate carrying tet(X4) originating from black kites. A combination of short- and long-read sequencing strategies showed that tet(X4) was located on a broad host range IncFII plasmid family in K. pneumoniae isolates whereas on an IncFII-IncFIB hybrid plasmid in E. coli. We also found an integrative and conjugative element ICEKp2 in K. pneumoniae isolate KP8336. We demonstrate the first description of tet(X4) gene in the WHO critical-priority pathogen K. pneumoniae among wild birds. The convergence of tet(X4) and virulence associated ICEKp2 in a wild bird with known anthropogenic contact should be further investigated to evaluate the potential epidemiological implications. The potential risk of global transmission of tet(X4)-positive K. pneumoniae and E. coli warrant comprehensive evaluation and emphasizes the need for effective mitigation strategies to reduce anthropogenic-driven dissemination of AMR in the environment.

antimicrobial-resistant bacteria/genes, likely from foraging in anthropogenically impacted areas including both landfills and WWTPs [7][8][9] , the role of wild birds in the dissemination of clinically relevant AMR needs further investigation 10 .Several studies have suggested that wild birds could be competent vectors of AMR and potentially disperse AMR through their movements 8,11,12 .Tigecycline is regarded as the last resort antibiotic in the clinical management of infections associated with MDR bacteria, particularly carbapenem and colistin-resistant Enterobacteriaceae.The recently discovered tet(X4) gene on plasmid confers resistance to tigecycline and has been found mainly in E. coli isolated from various sources, including humans, animals, and the environment 13 .The ability of the tet(X4) gene to be carried on various plasmid types, including hybrid plasmids, can facilitate its spread among bacterial populations and contribute to the emergence and spread of tigecycline-resistant bacteria.
Several studies have reported the presence of tigecycline-resistant E. coli carrying the tet(X4) gene in wild birds 17,18 , which raises concerns due to its potential spread in the environment.Additionally, this may lead to the transfer of tet(X4) to other bacteria, including those that cause human and animal infections.In this study, we investigated the prevalence and molecular characteristics of tet(X4) in K. pneumoniae and E. coli isolates from wild birds in Pakistan and further described the tet(X4)-harboring plasmids.

Sample collection and bacterial isolates
In this cross-sectional study, faecal droppings of 150 wild birds with known anthropogenic interaction (n = 100 from black kites and n = 50 from house crows) were collected from various public parks in Faisalabad, Pakistan during June 2022.A single fresh isolated faecal dropping sample was taken from an individual bird using sterile charcoal swabs and transported to the lab for microbiological examination.To prevent contamination, only the top surface of each dropping was swabbed, avoiding contact with the ground beneath.In addition, we confirmed the origin of the avian species by the direct visual observation of crows and kites in a public park.Collected samples were cultivated on Simmons citrate agar supplemented with amoxicillin and myo-inositol at concentrations of 10 µg/mL and 10%, respectively for the isolation of Klebsiella spp. 19.
For the isolation of E. coli, UTI ChromoSelect agar (Merck, Darmstadt, Germany) was used.For the isolation of tigecycline-resistant colonies, sub-culturing of both E. coli and Klebsiella spp. was performed on UTI ChromoSelect agar supplemented with tigecycline (4 µg/mL).All the phenotypically resistant isolates were confirmed for species using API 20E biochemical strips (bioMérieux, Marcy l'Etoile, France).For the detection of tet(X4)-positive E. coli and K. pneumoniae isolates, PCR was performed using primers described earlier 20 .Briefly tet(X4)-gene was amplified using primer pairs tetX4-F (5ʹ-CCG ATA TTC ATC ATC CAG AGG-3ʹ) and tetX4-R (5ʹ CGC TTA CTT TTC CAA GAC TTACC-3ʹ) as forward and reverse primers with 32 cycles of denaturation at 95 °C for 30 s; annealing at 55 °C for 30 s; extension at 72° for 30 s 20 .

Conjugation experiments
Conjugation assays were conducted to investigate the transferability of the tet(X4) positive isolates with sodium azide resistant E. coli J53 as the recipient strain.Transconjugants were selected on MacConkey agar containing (4 µg/mL) tigecycline combined with 100 µg/mL sodium azide.Subsequent carriage of tet(X4) bearing plasmids in the original parental strains and corresponding transconjugants was confirmed by PCR.

Whole-genome sequencing and bioinformatics analysis
The total genomic DNA of isolates from overnight cultures was prepared using MagnaPure compact total NA kit according to the manufacturer's instructions (Roche, Sweden).Library preparation was performed with the Illumina Nextera XT kit (Illumina, USA).Libraries were verified with the bioanalyzer high sensitivity DNA method (Agilent, USA).Paired-end sequencing (2 × 250 bp) of genomic DNA using a V3 run kit (Illumina) was performed on a MiSeq instrument (Illumina, San Diego, CA, United States).Short-read Illumina raw sequence reads were de novo assembled into contigs using SPAdes 21 and contigs less than 500 bp were discarded.Analysis of multilocus sequence typing (MLST), acquired resistance genes, and plasmid replicons were conducted by the online tools MLST, ResFinder, and PlasmidFinder at the web service of Center for Genomic Epidemiology (http:// www.genom icepi demio logy.org/.Kleborate, which was designed specifically for K. pneumoniae, was used to identify virulence factors and ICEKp structures, and further determine the sequence types (STs) of K. pneumoniae 22 .
To explore the evolutionary relationship of tet(X4)-positive K. pneumoniae between this study and other isolates in the NCBI database, all the tet(X4)-positive K. pneumoniae isolates were retrieved and downloaded from the NCBI Pathogen detection database.The isolates were retrieved with the search criteria ' AMR_genotypes: tet(X4)' and 'Organism_group: Klebsiella pneumoniae' as of March 2023.The draft assembled contigs were annotated using Prokka 23 , and then applied for phylogenetic analysis using Roary 24 and FastTree 25 based on single nucleotide polymorphisms (SNPs) of core genomes.The phylogenetic tree was visualized by iTOL (https:// itol.embl.de/ itol.cgi) 26 .
Genomic DNA was extracted using MagAttract HMW DNA kit according to the manufacturer's instructions (Qiagen, Sweden) and was then subjected to long-read sequencing to obtain the complete sequences 27,28 .Library preparation and sample barcoding were performed using Rapid sequencing gDNA-barcoding chemistry and protocol (Oxford Nanopore Technologies, UK, SQK-RBK110.96, version RPK_9126_v110_revK_24Mar2021).Sequencing was performed using MinION™ MK-1B with FLO-MIN106 R9.4.1 flow cell and high-accuracy basecalling with read filtering out at Q score < 9 and trimming of barcodes, MinKnow 22.03.6 and Guppy 6.0.7.www.nature.com/scientificreports/Short-read Illumina data and long-read Nanopore data were subjected to perform de novo assembly by Unicycler with the hybrid strategy.The rapid annotation website server (https:// rast.nmpdr.org/ rast.cgi) was then used to annotate the complete genome sequences 29 .Circular comparisons between tet(X4)-bearing plasmids and homologous plasmids available in the NCBI database were performed using the BRIG tool 30 .To visualize the genetic comparison features of ICEKp2, Easyfig was used to generate linear comparison figures 31 .

Identification of tet(X)-positive isolates and transferability
We identified two K. pneumoniae isolates (KP8333 and KP8336) and one E. coli isolate (EC8331) originating from black kites (Milvus migrans) with phenotypic resistance to tigecycline.All three isolates were also found to be positive for the carriage of tet(X4) gene and successfully transferred the tet(X4) gene into host E. coli J53 by conjugation.

The ICEKp structure in KP8336
The boundaries of ICEKp in KP8336 was identified by the 17 bp direct repeats (5ʹ-CCA GTC AGA GGA GCCAA-3ʹ) formed upon integration (Fig. 1B).Comparative analysis indicated that the ICEKp structure in KP8336 was one ICEKp2 variant, a 63 kb chromosomal island.The ICEKp2 structure mainly included the P4-like integrase gene int, the ybt locus (29 kb), and the sequence (14 kb) encoding the virB-type IV secretion system (T4SS) that is responsible for DNA transfer (Fig. 1B).In addition, the chromosome flanking ICEKp2 in KP8336 exhibited extremely high homology with the chromosome of K. pneumoniae 2485STDY5477980 isolated from human in UK (Fig. 1B).

Discussion
In this study, we isolated two tet(X4)-positive K. pneumoniae and one tet(X4)-positive E. coli isolate from black kites (Milvus migrans) in Faisalabad, Pakistan and performed a comparative genomic analysis on these isolates.Until now, only a few studies have reported the existence of tet(X4)-positive K. pneumoniae in human 16 , animal, and food specimens 32 predominantly from China.This is the first report of the presence of tet(X4)-positive K. pneumoniae in Pakistan indicating the spread of tet(X4) beyond E. coli.Black kite is an abundant bird and an opportunistic and scavenging feeder providing ecosystem functions such as nutrient cycling and pest control etc.In Pakistan, black kites frequently visit anthropogenic affected areas such as landfills, food-producing animals and agricultural land 33 .One more indigenous reason for anthropogenic interaction is that a fraction of the population from a dominant religious group believes in giving pieces of flesh (cow/goat/chicken) as a means of warding off calamities and hardships 34 .Recent findings suggest a link between certain wild bird species and the   acquisition of clinically relevant AMR.Notably, black kites have been reported to harbor carbapenem-resistant NDM-5-producing E. coli, in Pakistan 7 .Our finding of tet(X4)-positive K. pneumoniae in this wild bird species along with findings from previous studies is an indication that plasmid-mediated tigecycline resistance has the potential to disseminate within the one health framework like previously described with both ESBLs and carbapenem resistance genes 7,13,17,18,32,35 .Wild birds can acquire clinically relevant MDR bacteria, likely from foraging in anthropogenically impacted areas as reported earlier 36,37 .Tigecycline is rarely used in human medicine and is not used in food animals in Pakistan.However, plasmid-mediated tet(X4)-positive E. coli have been reported both from clinical and non-clinical settings 16,18 , which could be linked to excessive use of early generations of tetracycline antibiotics in food animals as proposed by several researchers 15,38 .However, the link between the long-term use of tetracyclines in food animals and the emergence of tigecycline resistance in bacteria needs to be ascertained.Genome data found that both the K. pneumoniae isolates have different STs and showed no clonal relationship with the global tet(X4)-positive K. pneumoniae strains in the NCBI database indicative of genetic diversity.Additionally, a single tet(X4)-positive E. coli isolate belonged to ST746 which has been previously associated with carbapenem resistance in Korea 39 and China 40 .It has been suggested that mobile genetic elements, not the clones, play an important role in tet(X4) transmission 32 .We found an integrative and conjugative elements ICEKp2 in K. pneumoniae KP8336 which carried ybt locus encoding yersiniabactin.It has been reported that ICEKp variants could form an extrachromosomal circular intermediate and integrate into the chromosomes of recipient cells 41,42 .These results suggest that the formation of tet(X4)-positive hypervirulent K. pneumoniae KP8336 may be due to the horizontal transfer of ICEKp2 and IncFII(29) plasmid carrying tet(X4).The finding of ICEKp2 in K. pneumoniae from wild birds with known anthropogenic interaction is concerning.K. pneumoniae with ICEKp2 has been associated with clinical outbreaks 16 and therefore, further investigative studies are important to be able to identify dissemination routes of AMR within the one health context.Additionally, mitigation efforts should be encouraged for already identified anthropogenic-driven AMR dissemination through waste, sewage and industrial pollution.
Hybrid genome assembly of E. coli EC8331 revealed that tet(X4) was located on a large MDR hybrid plasmid (~ 178 kb) IncFII-IncFIB (AP001918) plasmid harbouring additional resistance genes for quinolones, fosfomycin, macrolides, aminoglycosides and β-lactams.The hybrid plasmid pEC8331-tetX has large repeat structures surrounded by IS26, which has previously been reported to mediate tandem multiplication of genes within plasmids 43 .In addition, tet(X4) was flanked by two copies of ISCR2, forming a structure ISCR2-hp-abh-tet(X4)-ISCR2, which was found in several tet(X4)-positive isolates 20,44 .In previous studies from Pakistan, IncFII was the most common tet(X4) bearing plasmid ranging from 66 to 119 kbp in size 15,18 .tet(X4) bearing hybrid plasmids are being increasingly reported from China and are considered a new threat 41 .Therefore, the emergence of novel MDR hybrid plasmids in Pakistan is a serious concern because of their ability to contribute to the resistance and virulence genes co-translocation and demands continuous surveillance of AMR.Genomic comparison of tet(X4)-positive IncFII plasmid from K. pneumoniae KP8333 showed it has a similar genetic environment as that of E. coli isolated in this study and of chicken and human origins reported earlier in Pakistan 15,45 .These results indicate that E. coli and K. pneumoniae are important host bacteria of tet(X4)-bearing IncFII plasmids in Pakistan.

Conclusion
The prevalence and molecular features of the tet(X4) positive bacteria in wild birds demonstrate that this gene has disseminated within the One Health framework and is yet an example of wild birds as potential carriers of novel plasmid-mediated resistance genes together with hypervirulent traits.This emphasizes the need for mitigation strategies for anthropogenic-driven dissemination of AMR in the environment.
As part of a comprehensive One Health strategy, we advocate for increased environmental AMR surveillance.To effectively monitor these efforts, we propose more detailed studies on wildlife and their interactions with known anthropogenic sources of AMR.By leveraging animal movement data from GPS telemetry, we can gain valuable insights into dynamics of AMR at the human-wildlife interface.Obvious mitigation strategies would be to expanding already existing interventions aimed at reducing AMR dissemination targeting landfills and wastewater treatment plants.Future strategies might also include reducing wildlife access to identified point sources of AMR.
A better understanding of the human-animal-wildlife interface will guide the development of evidence-based and effective One Health interventions that ultimately can reduce the AMR crisis. https://doi.org/10.1038/s41598-024-59201-5

Figure 1 .
Figure 1.Phylogenetic tree of tet(X4)-positive K. pneumoniae strains and the ICEKp2 structure in KP8336.(A) Phylogenetic tree of 34 tet(X4)-positive K. pneumoniae strains, including two isolates in this study and 32 K. pneumoniae strains in the NCBI Pathogen detection database.The blue squares indicate the presence of virulence genes.(B) Alignment of the virulence-encoding region carried by KP8336, ICEKp2 structure in K. pneumonia 2485STDY5477980 and other chromosome regions of K. pneumonia 2485STDY5477980 (ERR314530).

Figure 2 .
Figure 2. Circular comparison of tet(X4)-bearing plasmid in EC8331 and other similar plasmids in NCBI database.The pEC8331-tetX in this study were compared with plasmids pCTXM-2271, pTEM, and pPK8261-tetX in the NCBI database.The outermost circle indicates the plasmid pEC8331-tetX with genes annotated.

Figure 3 .
Figure 3. Circular comparison of tet(X4)-bearing plasmid in KP8333 and other similar plasmids in NCBI database.The pEC8331-tetX in this study were compared with plasmids pPK8277-tetX, pPK5074-tetX, and pKP120-CTX-M-125 in the NCBI database.The outermost circle indicates the plasmid pKP8333-tetX with genes annotated.

Figure 4 .
Figure 4. Circular comparison of tet(X4)-bearing plasmid in KP8336 and other similar plasmids in NCBI database.The pEC8331-tetX in this study were compared with plasmids pPK8241-tetX, pRHB34-C05_2, and pJUNP054 in the NCBI database.The outermost circle indicates the plasmid pKP8336-tetX with genes annotated.

Table 1 .
Characteristics of three tet(X4)-carrying isolates recovered from birds in Pakistan.